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| United States Patent |
5,552,295
|
|
Stanker
, et al.
|
September 3, 1996
|
Monoclonal antibodies to bovine haptoglobin and methods for detecting
serum haptoglobin levels
Abstract
Hybridoma cell lines have been generated which produce and secrete
monoclonal antibodies which selectively bind bovine haptoglobin, either in
its native state or bound to hemoglobin and/or albumin. These hybridomas
may be obtained by using as an immunization agent or immunogen, bovine
haptoglobin that has been complexed to hemoglobin and optionally albumin.
Total bovine haptoglobin, including both native and bound to hemoglobin
and/or albumin, present in biological samples may be detected and
quantified by contacting the sample with the antibodies to form a bovine
haptoglobin/antibody immunocomplex when the bovine haptoglobin is present,
which immunocomplex may then be detected. The monoclonal antibodies may
also be incorporated into kits for the detection and quantification of
bovine haptoglobin.
| Inventors:
|
Stanker; Larry H. (College Station, TX);
Sheffield; Cynthia L. (College Station, TX);
DeLoach; John R. (College Station, TX)
|
| Assignee:
|
The United States of America as represented by the Secretary of (Washington, DC)
|
| Appl. No.:
|
205669 |
| Filed:
|
March 2, 1994 |
| Current U.S. Class: |
435/7.92; 435/337; 435/975; 436/518; 436/548; 530/388.1 |
| Intern'l Class: |
G01N 033/53; G01N 033/543; C07K 016/18; C12N 005/20 |
| Field of Search: |
436/518,548
530/392,385,388.1
435/975,240.27,7.92,975
|
References Cited
Other References
Busby and Travis, 1978, Comp. Biochem Phys. vol. 60B pp. 389-396 "Structure
and Evolution of Artiodactyla Haptoglobins".
Nakamura et al, Enzyme Immuroassays: Heterogeneous and Homogeneous system:
in Handbook of Experimental Immunology. 1986 Blackwell Scientific pub.
vol. 1, pp. 27.1-27.2.
"Immunogenicity and Antigen Structure in: Fundamental Immunology" Paul. ed.
1989 Raven Press, NY. pp. 177-178.
Katnik et al, 1989, Hybridoma V 8 No. 5 pp. 551-560. "Monoclonal Antibodies
Against Human Haptoglobin".
Eckersall and Connor, 1990 comp. Biochem. Physiol. vol. 96B No 2 pp.
309-314. "Plasma Haptoglobin in cattle . . . ".
Campbell, "Monoclonal Antibody and Immunosensor Technology in: Laboratory
techniques . . . " 1991, Elsevier Science Pub. NY, vol. 23, p. 45.
Katnik et al., 1993, Archivum Immunological and Therapial Experimentalis
vol. 41 pp. 105-109. "Quantitation of Human Heptoglobin by Elisa System .
. . ".
|
Primary Examiner: Scheiner; Toni R.
Assistant Examiner: Duffy; Patricia A.
Attorney, Agent or Firm: Silverstein; M. Howard, Deck; Randall E., Fado; John D.
Claims
We claim:
1. A hybridoma cell line which produces and secretes monoclonal antibodies
which specifically bind to bovine haptoglobin at non-hemoglobin binding
sites.
2. The hybridoma cell line of claim 1 wherein said monoclonal antibodies
specifically bind to bovine haptoglobin at non-hemoglobin and non-albumin
binding sites.
3. The hybridoma cell line of claim 1 which is produced using an
immunization preparation comprising a complex of bovine haptoglobin bound
to hemoglobin.
4. The hybridoma cell line of claim 3 wherein said immunization preparation
further comprises albmuin bound to said complex and said monoclonal
antibodies specifically bind to bovine haptoglobin at non-hemoglobin and
non-albumin binding sites.
5. A monoclonal antibody produced by the hybridoma cell line of claim 1.
6. A monoclonal antibody produced by the hybridoma cell line of claim 2.
7. A method for detecting or quantifying bovine haptoglobin in a biological
sample comprising:
(a) contacting said sample with the monoclonal antibody of claim 5 to form
a bovine haptoglobin/antibody immunocomplex when bovine haptoglobin is
present, and
(b) detecting the presence or amount of said immunocomplex.
8. The method of claim 7 wherein said sample is selected from the group
consisting of plasma, serum and whole blood.
9. The method of claim 7 wherein said contacting step comprises:
(1) providing a solid substrate;
(2) incubating said sample with said solid substrate to coat said substrate
with any bovine haptoglobin in said sample;
(3) rinsing said substrate;
(4) Incubating the bovine haptoglobin coated substrate from (2) with said
monoclonal antibody; and
(5) rinsing said substrate;
and wherein said detecting step comprises:
(4) detecting any monoclonal antibody bound to said substrate; and
(5) determining the presence or amount of bovine haptoglobin in said
sample.
10. A method for detecting or quantifying bovine haptoglobin in a
biological sample comprising:
(a) contacting said sample with the monoclonal antibody of claim 6 to form
a bovine haptoglobin/antibody immunocomplex when bovine haptoglobin is
present, and
(b) detecting the presence or amount of said immunocomplex.
11. The method of claim 10 wherein said sample is selected from the group
consisting of plasma, serum and whole blood.
12. A kit for the detection or quantification of bovine haptoglobin in a
biological sample comprising a container including the monoclonal antibody
of claim 5.
13. A kit for the detection or quantification of bovine haptoglobin in a
biological sample comprising a container including the monoclonal antibody
of claim 6.
14. A method for preparing hybridoma cell lines which produce and secrete
monoclonal antibodies which specifically bind to bovine haptoglobin at
non-hemoglobin binding sites comprising:
(a) immunizing a mammal with an immunization preparation comprising a
complex of bovine haptoglobin conjugated to hemoglobin;
(b) recovering antibody-producing cells from said mammal;
(c) fusing said antibody-producing cells with cells of a continuously
replicating cell line to form hybrid cells;
(d) screening said hybrid cells for the production of monoclonal antibodies
which specifically bind to bovine haptoglobin at non-hemoglobin binding
sites; and
(e) cloning said hybrid cells from (d) producing said antibodies.
15. The method of claim 14 wherein said mammal is a mouse.
16. The method of claim 14 wherein said immunization preparation further
comprises albumin conjugated to said complex.
17. The hybridoma cell lines of claim 1 which is produced by using an
immunization preparation comprising a complex of bovine haptoglobin
conjugated to hemoglobin, and screened for the production of monoclonal
antibodies which specifically bind to bovine haptoglobin at non-hemoglobin
binding sites.
18. The hybridoma cell line of claim 2 which is produced by using an
immunization preparation comprising a complex of bovine haptoglobin
conjugated to hemoglobin and albumin, and screened for the production of
monoclonal antibodies which specifically bind to bovine haptoglobin at
non-hemoglobin and non-albumin binding sites.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies
produced therefrom which may be used to detect haptoglobin.
2. Description of the Prior Art
Haptoglobin is a macromolecular glycoprotein which is the major acute phase
reactant in cattle and other ruminants [Bremner, 1964, Aust. J. Exp. Biol.
Med. Sci., 42:643-656; Goodger, 1970, Clin. Chim. Acta., 29:429-435;
Blakeslee and Stone, 1971, Vox Sang., 21:175-182; Spooner and Miller,
1971, Vet. Rec., 12:2-4; Putnam, 1975, Haptoglobin, In: The Plasma
Proteins, Structure, Function, and Genetic Control, 11, Putnam (ed.),
Academic Press, New York, pp. 1-50; Blackshaw, 1979, NZ Vet. J.,
27:103-105; Eckersall and Conner, 1990, Comp. Biochem. Physiol.,
96B:309-314; and Morimatsu et al., 1991, J. Biol. Chem., 266:11833-11837].
Biochemical studies have shown that bovine haptoglobin is composed of two
chains, an alpha-chain and a beta-chain. The reported molecular weights of
the chains has varied among investigators, with the alpha-chain having
been described as 16 and 23 KDa, and the beta-chain having been described
as 40 and 35 KDa [Morimatsu et al., ibid, Eckersall and Conner, ibid]. Two
alpha-chains and two beta-chains are linked by a disulfide bond, forming
the basic tetrameric subunit of the compound. In biological material such
as blood, haptoglobin binds to hemoglobin, forming a stable complex
therewith [Putnam, ibid; Morimatsu et al., ibid]. Eckersall and Conner
(ibid) have also reported that haptoglobin is associated with albumin in
bovine plasma. The basic tetrameric subunit and albumin associate into
polymers of varying molecular weight. Eckersall and Conner have reported
molecular weights of 670 KDa and Morimatsu et al. have reported variable
molecular weights up to 2000 KDa.
As an acute phase reactant, haptoglobin serum levels become greatly
elevated immediately following injury or infection. Clinically, less than
5% of healthy cattle have measurable levels [10 mg % hemoglobin binding
capacity (HbBC)] of haptoglobin in their serum (Bremner; Goodger;
Blakeslee and Stone; Spooner and Miller; Blackshaw; and Eckersall and
Conner, all ibid). However, a rapid increase in the serum haptoglobin
level occurs following infection, inflammation or trauma, with serum
levels exceeding 100 mg % HbBC of haptoglobin within 3 to 4 days following
trauma (Spooner and Miller, ibid). Haptoglobin therefore represents a
nonspecific, highly sensitive indicator of disease or tissue damage in
cattle [Liberg, 1977, Acta Vet. Scand., 18:335-348; Dinarello, 1984,
Induction of Acute Phase Reactants by Interleukin-1, IN: Advances in
Inflammation Research, 8, Weissman (ed.), Raven Press, New York, pp.
203-225; Conner et al., 1986, Res. Vet. Sci., 41:126-128; and Eckersall
and Conner, 1988, Vet. Res. Commun., 12:169-178].
Considerable research has dealt with illuminating the conditions that
stimulate the production of haptoglobin and the in vivo time course of
haptoglobin production [Blakeslee and Stone; Spooner and Miller; Liberg;
Blackshaw; Dinarello; Conner et al. 1986; and Eckersall and Conner 1988,
all ibid; and Conner et al., 1986 Prot. Biol. Fluids, 34:509-512; Conner
et al., 1988, Res. Vet. Sci., 44:82-88; Conner et al., 1989, Res. Vet.
Sci., 47:203-207; Erskine and Ridell, 1990, The Acute Phase Response
During Experimental Escherichia coli Mastitis, In: The International
Symposium on Bovine Mastitis, Sep. 13-16, 1990, Indianapolis, Ind., pp.
60-63; and Makimura and Usui, 1990, Jpn. J. Vet. Sci., 52(6):1245-1250].
Additional studies have focused on the purification and biochemical
characterization of haptoglobin [Owen et al., 1960, J. Clin. Pathol.,
13:163-164; Bremner, ibid; Goodger 1970, ibid; Goodger, 1972, Aust. J.
Exp. Biol. Med. Sci., 50:11-20; Javid and Liang, 1973, J. Lab. Clin. Med.,
82:991-1002; Spooner, 1973, Res. Vet. Sci., 4:90-96; Makimura and Suzuki,
1982, Jpn. J. Vet. Sci., 44:15-21; Osada, 1985, Acta Biochim. Pol.,
32:225-233; Eckersall and Conner 1990, ibid; Morimatsu et al., ibid;
Skinner et al., 1991, Vet. Rec., 128:147-149; and Yoshino et al., 1992,
Am. J. Vet. Res., 53(6):951-956].
There exists a need for an indicator of preclinical illness in cattle to
reduce the number of carcasses condemned after slaughter. However, the use
of serum haptoglobin levels as this diagnostic tool has been hampered by
the lack of a simple, rapid, and sensitive assay for determining
haptoglobin concentrations. The current method used to quantify
haptoglobin is a colorimetric procedure based upon the difference in
peroxidase activity between free hemoglobin and the hemoglobin-haptoglobin
complex (Owen et al.; and Makimura and Suzuki, both ibid). Unfortunately,
this is a cumbersome assay, and the accuracy of the method is dependent
upon the quality of the sample, particularly the collection of plasma that
is free of hemoglobin contamination.
SUMMARY OF THE INVENTION
We have now discovered hybridoma cell lines which produce and secrete
monoclonal antibodies which selectively bind haptoglobin, either in its
native state or bound to hemoglobin and/or albumin. We have unexpectedly
found that these hybridomas may be obtained by using as an immunization
agent or immunogen, haptoglobin that has been complexed to hemoglobin and
optionally albumin. Total haptoglobin, including both native and bound to
hemoglobin and/or albumin, present in biological samples may be detected
and quantified by contacting the sample with the antibodies to form a
haptoglobin/antibody immunocomplex when the haptoglobin is present, which
immunocomplex may then be detected. The monoclonal antibodies may also be
incorporated into kits for the detection and quantification of
haptoglobin.
It is an object of this invention to provide hybridoma cell lines that
produce and secrete high affinity monoclonal antibodies which selectively
bind haptoglobin, and which are effective for detecting free haptoglobin
and haptoglobin conjugated to hemoglobin and/or albumin.
Another object of this invention is to provide immunoassay methods for the
measurement of haptoglobin in biological samples as an indicator of
preclinical illness in animals, particularly bovine.
A further object is to provide kits useful for the assay of bovine
haptoglobin which include the monoclonal antibodies described herein.
Other objects and advantages of this invention will become readily apparent
from the ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an SDS-PAGE gel of test and control plasma retentates.
FIG. 2 shows a cellulose acetate electrophoresis gel of test and control
plasma retentates.
FIGS. 3A and 3B show typical ELISA results of menoclonal antibody analysis
on Microtiter plates coated with test plasma retentate, control plasma
retentate, and bovine hemoglobin.
FIG. 4 shows an immunoblot of a thin layer agarose electrophoresis gel of
test and control plasma retentates.
FIG. 5 shows a typical ELISA standard curve for haptoglobin. A linear
regression curve fit is shown.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention we have created hybridoma cell lines that
produce monoclonal antibodies which selectively bind haptoglobin not only
in its native state but also haptoglobin bound to hemoglobin and/or
albumin. Without being limited thereto, the invention is particularly
suited to the production of monoclonal antibodies binding to haptoglobin
of ruminants such as goats, sheep, deer and especially bovine. The
antibodies may be used to rapidly and accurately detect and quantify
haptoglobin, providing an indicator of preclinical illness in animals such
as cattle and reducing the number of carcasses condemned after slaughter.
Serum samples are often contaminated with hemoglobin that binds to
haptoglobin, interfering with traditional analytical methods. Although the
development of specific antibodies would be desirable for the detection of
haptoglobin in biological samples, here again the presence hemoglobin may
interfere and lead to inaccurate results. For example, the
antigen-antibody binding of many monoclonal antibodies generated to pure
haptoglobin may be sterically inhibited or prevented by the presence of
hemoglobin contaminants bound to the haptoglobin molecule. Depending upon
the degree of hemoglobin contamination and its inhibition of antibody
binding, this could lead to significant errors or false negative results.
We have discovered that this problem may be overcome by using haptoglobin
which is complexed to hemoglobin as an immunization agent for the
preparation of hybridomas. The monoclonal antibodies produced by these
hybridomas bind to haptoglobin at non-hemoglobin binding sites on the
molecule, and consequently bind to both free haptoglobin and haptoglobin
that is complexed to hemoglobin in the sample. The antibodies therefore
provide an accurate tool for the measurement of total haptoglobin in
biological samples that is free from interference by hemoglobin
contaminants.
In a particularly preferred embodiment, the immunization agent also
includes albumin conjugated to the haptoglobin together with hemoglobin.
Although not essential, inclusion of albumin is highly desirable to
generate hybridoma cell lines producing monoclonal antibodies that bind to
native haptoglobin as well as haptoglobin bound to hemoglobin and/or
albumin. As noted hereinabove, albumin has also been reported bind with
haptoglobin in biological samples such as blood. By use of a
haptoglobin-hemoglobin-albumin complex as the immunization agent,
monoclonal antibodies that bind to haptoglobin at non-hemoglobin and
non-albumin binding sites may be produced. Not only would the binding of
these monoclonal antibodies be free of interference by hemoglobin
contaminants, they would also be free from interference by albumin as
well.
Preparation of the hybridomas may be accomplished using conventional
techniques such as described by Kohler and Milstein [Nature, 256:495-497
(1975)], Koprowski et al. [U.S. Pat. No. 4,196,265], Wands [U.S. Pat. No.
4,271,145], or Stanker et al. [U.S. patent application Ser. No.
08/081,591, filed Jun. 23, 1993], the contents of each of which are
incorporated by reference herein. Generally, the process of preparation
comprises the steps of immunizing an animal with the antigen of interest,
recovering splenocytes or lymphocytes from the animal, fusing the
splenocytes or lymphocytes with continuously replicating myeloma cells to
produce hybrid cells, and screening the resultant hybrid cells for the
production of antibodies to the antigen.
The method of preparing the hybridomas comprises the following steps:
Immunogen. The immunizing agent is constructed by complexing haptoglobin
with hemoglobin and optionally albumin. In a preferred embodiment, the
immunogen is derived from the plasma of subject traumatized animals,
particularly bovine. The manner of traumatizing the animal is not
critical, and a variety of treatments effective for eliciting haptoglobin
production which are known in the art may be used herein. Without being
limited thereto, preferred traumatizing treatments include humane exposure
to non-lethal levels of inflammatory agents or noxious chemicals,
particularly turpentine. Following treatment, plasma from the subject
animals may be recovered, usually about 3 to 4 days after treatment.
Rather than isolating the haptoglobin from the plasma in pure form,
ideally, the haptoglobin is only partially purified, so as to maintain it
in association with hemoglobin and albumin from the original sample.
Without wishing to be limited to theory, this partial purification would
also allow the haptoglobin to be maintained in association with any other
as yet currently unknown binding proteins. To ensure complex formation,
hemoglobin may be optionally added to the plasma sample. The manner of
recovering the haptoglobin-hemoglobin complex from the plasma is not
critical, and a variety of techniques may be used including, but not
limited to, dialysis or diafiltration to remove low molecular weight
components (below 300 KDa). The resultant partially purified
haptoglobin-hemoglobin complex may be used directly as an immunogen.
In an alternative embodiment, the immunogen may be readily prepared from
pure haptoglobin. Conditions for the complex formation are not critical;
haptoglobin may be contacted with hemoglobin and preferably albumin in any
suitable solvent, such as physiologically buffered saline or water, with
spontaneous complex formation.
Immunization. To generate antibody-producing splenocytes or lymphocytes, an
immunizing preparation comprising the haptoglobin-hemoglobin complex is
injected into an immunologically competent animal. The preparation may
also contain other proteins, although partially purified compositions of
the conjugate in a pharmaceutically acceptable carrier are preferred.
Without being limited thereto, rats and particularly mice are preferred
animals for immunization because of ease of handling. Preparation of
hybridomas using splenocytes from these animals fused to a variety of
myeloma cell lines have been reported by many investigators.
Inoculations of the animal can be by various routes. A series of three
inoculations, generally at two week intervals, with a composition of the
complex in isotonic saline with RIBI adjuvant (Immunochem Research, Inc.,
Hamilton, Mont.) elicits good antibody response, and is preferred. The
skilled practitioner will recognize that other routes of administration,
immunization schedules, and carriers or adjuvants may be used.
Hybridization. Splenocytes or lymphocytes recovered from the immunized
animal are fused with continuously replicating tumor cells, such as
myeloma or lymphoma cells, cultured, and hybridoma cells selected using
techniques conventional in the art. Many continuously replicating tumor
cell lines are available which may be used as fusion partners with the
splenocytes. Without being limited thereto, preferred myeloma cells
include P3-NS1-K653, and particularly SP2/O.
Fusion and culture of the cells can be performed using conventional
techniques. In accordance with one well known effective procedure, the
splenocytes and myeloma cells are fused by exposure to polyethylene
glycol. Hybrid cells are selected by culture in
hypoxanthine-aminopterin-thymidine (HAT) medium, whereby unfused myeloma
cells are killed by HAT and splenocytes die out, leaving only the hybrid
cells. The resultant hybridomas are then grown in HAT or other suitable
culture medium and assayed for antibody production.
Screening. Samples of the supernatant culture fluid from the hybridomas are
screened for antibodies to haptoglobin. While the supernatants may be
screened using a plurality of techniques such as RIA, FIA and ELISA, in
accordance with the preferred embodiment, a modification of the
direct-binding ELISA (db-ELISA) is employed. Generally, solid substrates,
such as beads or the wells of a microtiter plate, which are coated with
haptoglobin or haptoglobin-hemoglobin complex, are used to bind
anti-haptoglobin antibody in the supernatants, and bound antibody is then
detected. Detection of bound antibody may be accomplished by addition of
enzyme-labeled anti-immunoglobulin antibodies followed by enzyme
substrate. Horse radish peroxidase and its substrate,
2,2'-azinobis-3-ethylbenthiazolinesulfonic acid (ABTS) are preferred
enzyme/substrate labels. However, it is understood that other
enzyme/substrate labels or non-enzyme labels such as radiolabels or
chromophores may also be used. The skilled practitioner will recognize
that in the event that this screening is conducted using substrates coated
with complexed haptoglobin (haptoglobin complexed with hemoglobin and/or
albumin), the antibodies should also be screened against a control, such
as hemoglobin and/or albumin, or control plasma from a non-traumatized
animal. This additional screen is necessary to ensure that only those
antibodies are selected which specifically bind haptoglobin, but not
hemoglobin, albumin or other serum proteins.
Cloning. Cloning of hybridomas which are positive for desired antibody
production can be carried out as soon as they are detected by any method
known in the art. Hybridomas having a positive response in the ELISA
screen are preferably expanded and subcloned one or more times by limiting
dilution to assure monoclonality.
The supernatant culture fluid from the cloned hybridomas may also be
screened to select for those producing antibodies having a high affinity
for haptoglobin. Affinity may be measured using a variety of well known
techniques, such as ELISA, RIA or equilibrium dialysis using labelled
haptoglobin.
Once hybridomas producing and secreting the desired anti-haptoglobin
antibodies are identified, large quantities of the antibody may be
produced in tissue culture using well-known techniques. Alternatively,
antibody may be produced within host animals, such as by ascites formation
in syngenic mice.
The monoclonal antibodies produced in accordance with this invention
possess high affinity for haptoglobin, allowing the rapid determination of
this acute phase reactant. The antibodies my be used in a variety of
conventional immmosorbent assays to detect and/or quantify haptoglobin in
unknown biological samples. Furthermore, immunoassay of haptoglobin with
the antibodies provides much greater sensitivity to haptoglobin, and
allows the detection of haptoglobin elevation in animals such as bovine
much earlier following infection or trauma, than conventional biochemical
methods.
Suitable assay procedures include but are not limited to RIA or ELISA,
although competitive inhibition ELISA or a direct-binding ELISA similar to
that used to screen the hybridomas is preferred. In the competitive
inhibition ELISA, a sample to be analyzed is incubated with the monoclonal
antibody for haptoglobin and a solid substrate coated with haptoglobin.
After incubation, the solid phase is drained and washed, and bound
antibody on the substrate is detected and percent inhibition calculated.
The concentration of haptoglobin in the sample may then be determined by
reference to a standard curve. A standard curve relating the percent
inhibition (amount of bound antibody) to haptoglobin concentration my be
constructed from assays using known levels of haptoglobin.
In another alternative embodiment, haptoglobin may be determined by a
competition ELISA such as described in Brandon et al. (U.S. Pat. No.
5,053,327, the contents of which are incorporated by reference herein)
using the monoclonal antibody of the invention attached to a solid
support. For example, the anti-haptoglobin antibody may be immobilized on
a solid support such as a bead or microtiter well. The unknown sample to
be analyzed (or analytical standards of haptoglobin) are then added with
enzyme or radiolabeled haptoglobin, and the amount of labeled haptoglobin
bound to the antibody is measured, using a substrate when the label is an
enzyme. The amount of haptoglobin in the sample is inversely proportional
to the amount of bound labeled haptoglobin. In yet another alternative,
the monoclonal antibody may be attached to a solid support for use in
conventional double-antibody sandwich ELISA procedures.
With any of the above-described assay formats, the monoclonal antibodies of
the invention may be incorporated into kits, alone or preferably together
with any other necessary reagents. A preferred kit for use herein
comprises a first container including the monoclonal antibody, a second
container including detection means effective for detecting bound
antibody, and a solid phase support having haptoglobin attached thereto.
Determination of haptoglobin in a variety of biological samples may be
conducted using the above-described assays. Without being limited thereto,
the assays are particularly advantageous for determining haptoglobin
levels in serum, plasma and whole blood.
In another application, the monoclonal antibodies may be incorporated into
sensors such as solid phase electronic devices for detection of
haptoglobin in sample materials.
The following examples are intended only to further illustrate the
invention and are not intended to limit the scope of the invention which
is defined by the claims.
EXAMPLE 1
Immunogen Production
Haptoglobin Induction. The immunogen preparation used for the development
of the monoclonal antibodies was derived from the plasma of
turpentine-treated cattle. Four purebred Hereford heifers (average body
weight (BW)=132 kg) were housed individually in indoor stalls, and fed a
diet of grass hay and a commercial range cube supplement. The heifers were
acclimated for 2 weeks to their diet and surroundings. The treatment and
control groups each contained two heifers. All heifers were anesthetized
by an intravenous injection of xylazine at a rate of 0.11 m/kg BW. When
the animal reached a light surgical plane of anesthesia, an injection site
located 6" caudal to the shoulder and 3" below the midline measuring
approximately 2".times.6" was shaved and cleaned by 3 washes with
Betadine, followed by a final wipe down with isopropyl alcohol. Each
treatment animal then received three 5 mL subcutaneous injections of
commercial grade turpentine approximately 2" apart. The control animal
received three 5 mL subcutaneous injections of sterile normal saline
approximately 2" apart. All animals were revived by an intravenous
injection of Tolazoline (300 mg/animal). Once alert, the heifers were
returned to their stalls and maintained as before. None of the animals
evidenced any signs of discomfort or required any specialized care. All of
the procedures involving the research animals were conducted as outlined
in protocol #91008 which was approved by the USDA, ARS Animal Care and Use
Committee in College Station, Tex. On days 2 to 5, post-treatment, 500 mL
of blood/animal were collected via jugular venipuncture into sterile
bottles containing 100 mL of anticoagulant citrate dextrose solution
(Sonofi Animal Health, Inc.). The erythrocytes were separated from the
plasma by centrifugation at 1000.times. g for 30 min. The plasma was
recovered and stored at -70.degree. C.
Haptoglobin Processing. Plasma from the treated animals was thawed and
delipidated by the addition of Seroclear (Cal Biochem, Inc., San Diego,
Calif.) according to the manufacturer's instructions. In some instances,
bovine hemoglobin was added to the delipidated, plasma and this material
was diafiltered using an Amicon Stirred Cell Chamber (Amicon, Inc.,
Danvers, Mass.) fitted with a 300 KDa molecular weight cutoff membrane.
The retentate was washed 3 to 4 times with 100 mL of phosphate buffered
0.9% saline (PBS; pH 7.4). After the final rinse and filtration, the
sample was resuspended in 10 mL of PBS, representing approximately a
10-fold concentration of the starting material. This retentate was
aliquoted and stored at -70.degree. C. until needed and is referred to as
the test plasma retentate (TPR). Plasma from the control animal was
processed similarly and is referred to as the control plasma retentate
(CPR).
Standard Curve Generation. The 4-day post-treatment test plasma (TP) was
diluted with control plasma as follows: 60% TP, 36% TP, 22% TP, 13% TP, 8%
TP, and 5% TP. The haptoglobin level was determined as the mg % HbBC
(hemoglobin binding capacity) using the differential peroxidase method of
Owen et al. (1960, J. Clin. Path., 13:163 -164), the contents of which are
incorporated by reference herein. These samples were then used in an ELISA
as described in Example 2 to generate a standard curve. The plasma samples
collected at 4-days post-treatment from turpentine treated test cows were
found to have a haptoglobin level of 104 mg % HbBC by the differential
peroxidase method. The plasma samples collected 4-days post-treatment from
the control cows were found to have no detectable level of haptoglobin.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Both TPR and CPR were
analyzed by SDS-PAGE using 4-15% precast TRIS-HCl gradient gels (BioRad,
Inc., Richmond, Calif.). Samples (50 .mu.L) were loaded and
electrophoresed at 50 milliamps constant current for 1 hour (Laemmli,
1970). The gels were stained with Coomassie R-250 Brilliant Blue (0.2%
Coomassie R-250 Brilliant Blue (w/v), 25% isopropyl alcohol, 10% acetic
acid) for 1 hour, then destained (in 25% isopropyl alcohol, 10% acetic
acid) until a colorless background was obtained.
SDS-PAGE analysis of the TPR revealed two bands not present in the CPR; the
beta-subunit of haptoglobin with a molecular weight of 38.3 KDa and the
alpha-subunit of haptoglobin with a molecular weight 22.5 KDa (arrows)
(FIG. 1, lanes 1 and 2). Both the TPR and CPR contained numerous other
proteins, primarily albmuin.
Thin Layer Agarose. Thin layer agarose gel electrophoresis was performed on
TPR, TPR with bovine hemoglobin added, and CPR using precast gels (Titan
Gel Serum Protein System, Helena Laboratories Inc., Beaumont, Tex.).
Samples (3 .mu.l) were loaded and electrophoresed at 120 volts for 15 min.
The gels were fixed in 100% methanol for 5 min., dried at 70.degree. C.
for 15 min., then stained and destained as described above.
Cellulose Acetate. Cellulose acetate electrophoresis was also conducted on
the plasma retentates. The cellulose acetate plates were soaked for 10 to
15 min. in Titan Gel High Resolution Protein Buffer (Helena Laboratories
Inc., Beaumont, Tex.) prior to sample application. A 10 g % solution of
stabilized human hemoglobin (Helena Laboratories Inc., Beaumont, Tex.) was
mixed in a 1:20 ratio with the plasma retentate samples and allowed to
stand at room temperature for 5 min. Samples were applied to the cellulose
acetate plate using the Super-Z applicator (Helena Laboratories Inc.,
Beaumont, Tex.) and electrophoresed at 180 volts for 20 min. After
electrophoresis, the plates were fixed and stained according to the
manufacturer's instructions (Helena Laboratories Inc., Beaumont, Tex.).
Results from our analysis of TPR and CPR for haptoglobin using the Helena
Cellulose Acetate system are shown in FIG. 2. The single band migration
pattern of hemoglobin is shown in lane 1. A mixture of human hemoglobin
and human haptoglobin type 2-2 was electrophoresed and showed the
characteristic two band pattern (lane 2). The lower band is excess
hemoglobin while the upper band is the hemoglobin-haptoglobin complex.
Electrophoresis of a mixture of TPR and human hemoglobin (lane 3) also
showed 2 bands, the lower hemoglobin band and an upper
hemoglobin-haptoglobin complex. In contrast, identical analysis of CPR
(lane 4) displayed only a single hemoglobin band. The cellulose acetate
gels were stained with 0-dianisidine, which is a stain specific for
hemoglobin.
The Helena cellulose acetate electrophoresis method used is a clinical
method for the evaluation of human haptoglobin (Valeri et al., 1965). A
similar cellulose acetate electrophoresis method was used by Goodger
(1970, ibid) to detect bovine haptoglobin. The electrophoretic conditions
used in the Helena cellulose acetate method were designed to give maximum
separation of the human hemoglobin-haptoglobin complex, which is quite
different from the bovine complex (Goodger, 1972, ibid; Eckersal et al.,
1988, ibid). For this reason, the bovine hemoglobin-haptoglobin complex,
though clearly present (FIG. 2, lane 3), does not give as sharp a
separation as does the human complex (FIG. 2, lane 2). Nevertheless, the
results of the cellulose acetate haptoglobin assay clearly demonstrated
that the TPR contains a hemoglobin binding protein (FIG. 2, lane 3) that
is not found in the CPR (FIG. 2, lane 4). This hemoglobin binding protein,
is by definition, haptoglobin (Bremner; Spooner and Miller; Goodger, 1972;
Osada; Eckersall and Conner, 1990; Makimura and Usui; and Morimatsu et
al., all ibid). Thus, the results of the SDS-Page and the Helena cellulose
acetate electrophoresis both confirm that the TPR, used as antigen to
immunize the mice, contained bovine haptoglobin.
EXAMPLE 2
Hybridoma Production
The use of a partially purified immunogen (TPR) from Example 1 allowed the
haptoglobin to be maintained in association with both albumin and
hemoglobin, thus enabling the selection of monoclonal antibodies (Mabs)
which can detect haptoglobin when bound to hemoglobin. However, the use of
a complex antigen necessitated a differential screening procedure to
ensure isolation of a haptoglobin specific Mab. These efforts yielded
three haptoglobin specific Mabs and one bovine hemoglobin specific Mab.
The protein concentration of the hemoglobin containing TPR was determined
using the Sigma Total Protein Colorimetric Endpoint Diagnostic kit (St.
Louis, Mo.). The hemoglobin-haptoglobin TPR was combined with RIBI
adjuvant (Immunochem Research, Inc., Hamilton, Mont.) to a final protein
concentration of 250 .mu.g/mL and was used to immunize BALB/c mice. The
mice were given three intraperitoneal (Ip) antigen injections (100
.mu.L/injection) at 14 day intervals. A final Ip injection was given five
days prior to fusion. Ten days after the second immunization, plasma was
collected from each mouse by retro-orbital bleeding. The mice were given a
subcutaneous injection of a 10.times. diluted solution of Fentanyl
Citrate-Droperidol (Innovar Vet, Pittmon-Moore, Washington Crossing, N.J.)
5 to 10 min. prior to bleeding.
The mouse with the highest serum titer for TPR, when screened against TPR
and CPR in an ELISA as described hereinbelow, was selected for fusion.
Hybridomas were generated by fusion to mouse myeloma cells as described by
Stanker et al. (1986, J. Immunol., 136(11);4147-4180), the contents of
which are incorporated by reference herein. The cell fusion product was
spread over 30, 96-well plates.
Screening. Eleven to fifteen days after fusion, the hybridomas were
screened for antibodies that were positive against test plasma (TPR) and
negative against control plasma (CPR) using an ELISA. At fourteen days
post-fusion, greater than 90% of the wells contained hybridomas.
Cells that had a positive response to the test plasma and a negative
response to the control plasma were transferred to 24-well plates,
expanded, and subcloned at least twice by limiting dilution to ensure
their monoclonal origin. Additional screening of all clones was conducted
on microtiter plates coated with TPR, CPR, and bovine hemoglobin.
ELISA. The ELISA technique described below was used for all screening and
subsequent testing of animal plasma. Microtiter plates (96-well) were
coated with 100 .mu.L of antigen (TPR, CPR or bovine hemoglobin in 50 mM
carbonate buffer pH 9.6) at a concentration of 100 ng total protein/well,
and dried overnight at 37.degree. C. Immediately prior to use, nonreacted
sites on the plates were blocked by adding 3% nonfat dry milk (NFDM) in
phosphate buffered saline pH 7.0 (PBS-7) and incubating the plate for 30
min. The blocking solution was then discarded and 100 .mu.L/well of the
anti-haptoglobin Mab, either in the form of neat cell culture media (used
in preliminary screens) or a 1:1000 dilution of purified antibody stock
(1.4 mg/mL in 1% NFDM PBS-7 solution)(AB), was added and the plate
incubated for 1 hour at 37.degree. C. After incubation, the
anti-haptoglobin Mab was discarded and the plates washed 5 times in a
0.05% TWEEN-20/deionized water detergent wash solution (DWS) and once in
deionized water. Goat anti-mouse IgG-whole molecule-peroxidase conjugate
(Sigma Inc., St. Louis, Mo.), diluted 1:500 with AB was then added to the
plate (100 .mu.L/well), and the plate incubated 1 hour at 37.degree. C.
and washed as described above. Finally, 100 .mu.L/well of substrate (0.8
mM [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] (ABTS), 0.001%
H.sub.2 O.sub.2, in a 0.1M citrate buffer pH 5.0) was added. The plate was
then incubated for 45 min. at room temperature, and the O.D. at 405 nm
recorded.
Eighty wells contained hybridomas that responded positively to the TPR and
negatively to the CPR. Cells from these wells were subcloned twice by
limiting dilution. Only 14 clones survived the subcloning process.
Subsequent characterization of these Mabs by ELISA revealed 12 clones that
bound TPR bat not CPR or bovine hemoglobin (anti-haptoglobin Mabs) and 2
clones that bound both TPR and bovine hemoglobin (anti-bovine Mabs). The 3
clones (designated Hap1, Hap2, Hap3) with the highest titer to TPR, and
the clone designated (BoHem1) with the highest titer to bovine hemoglobin
were selected for further examination. FIGS. 3A and 3B are a typical ELISA
titration showing the reactivity of Hap1 (Panel A) and BoHem1 (Panel B) on
the antigens indicated. The titration curves in FIG. 3A clearly show that
Hap1 bound the TPR (solid circle), but not CPR (open circle) or bovine
hemoglobin (solid square). In contrast, BoHem1 (FIG. 3b) bound the TPR
(solid circle) and bovine hemoglobin (solid square), bat not CPR (open
circle). Monoclonal antibody Hap1 was selected for assay development and
immunoblotting evaluations.
Hybridoma Isotyping. The four monoclonal antibodies were isotyped according
to manufacturer's recommendations, using the FisherBiotech (Fisher
Scientific, Pittsburgh, Pa.) isotyping kit. All were found to be
IgG1-kappa antibodies.
Immunoblotting. The antibodies were further characterized by immunoblotting
using the electrophoresis gel samples prepared as in Example 1 (SDS-PAGE,
Thin Layer Agarose, and Cellulose Acetate).
a. Thin Layer Agarose:
Following electrophoresis, the thin layer agarose gels (Titan Gel Serum
Protein System, Helena Laboratories, Inc., Beaumont, Tex.) were dried
overnight at 37.degree. C. without fixation. Immediately prior to
immunoblotting, the gels were blocked with 3% BSA PBS-7 for 30 min. The
gels were then incubated (1 hour at room temperature with gentle
agitation) in the following sequence of materials:
A) 8 mL of Hap1 Mab, either in the form of neat cell culture media, or a
1:1000 dilution in 0.05M Tris-HCl buffered saline pH 7.4 (TBS) of purified
antibody stock (1.4 mg/mL);
B) 8 mL of biotinylated anti-mouse antibody (Sigma Inc., St. Louis, Mo.)
diluted 1:1000 in TBS;
C) 8 mL of streptavidin-biotinylated-alkaline phosphatase conjugate (Sigma
Inc., St. Louis, Mo.) diluted 1:1000 in TBS. The gels were washed 5 times
with 8 mL of DWS after each incubation. The gels were then incubated at
room temperature without agitation for no longer than 30 min. in 8 mL of
color development reagent (5-bromo-4-chloro-3-indolylphosphate, BCIP;
p-nitro blue tetrazolium chloride, (NBT); Sigma Inc., St. Louis, Mo.).
b. SDS-PAGE:
Proteins from 4-15% SDS gradient gels were transferred onto Immobilon-P
transfer membrane (Millipore, Bedford, Mass.) in a semi-dry transfer cell
(BioRad Laboratories, Hercules, Calif.). The Bjerrum and Schafer-Nielsen
transfer buffer system recommended by the manufacturer (BioRad
Laboratories, Hercules, Calif.) was used for all transfers. The proteins
were then visualized following the same immunoblotting procedure described
above.
c. Cellulose Acetate:
Cellulose acetate plates were air dried at room temperature overnight. The
proteins were then visualized following the same immunoblotting procedure
described above.
d. Results:
Samples identical to those electrophoresed on the cellulose acetate (FIG.
2, lanes 1-4) were probed with Mab Hap1 (FIG. 2, lanes 5-8). Hap1 labeled
only a single protein (lane 7), corresponding in position to the bovine
haptoglobin-hemoglobin complex seen in lane 3. No immunoprecipitation
bands are observed in lane 5 (free Hemoglobin), lane 6 (human Haptoglobin
2-2), or lane 8 (CPR).
Shown in FIG. 4 is a thin layer agarose gel probed with Hap1. Numerous
proteins were observed in both the CPR and TPR lanes when stained with
Coomassie Brilliant Blue (FIG. 4, lanes 1-2). In contrast, when Hap1 was
used as a probe, only a singly protein band was labeled when TPR was
analyzed (lanes 3 and 6), and no protein bands were labeled when CPR was
analyzed (lane 4) or when bovine hemoglobin was analyzed (lane 5). The
location of the immunostained band (haptoglobin) in lane 3 corresponds to
a region in lane 2 (TPR) that is more heavily stained with Coomassie
Brilliant Blue than the corresponding region in lane 1 (CPR). The addition
of free hemoglobin to the test plasma prior to electrophoresis, resulted
in a shift of the immunostained band (lane 7) toward the cathode relative
to the position of the immunostained TPR band (lane 6) (Compare FIG. 4,
lane 6 with lane 7). Bremmer (ibid) using paper electrophoresis, reported
that bovine hapteglobin migrated to a location between alpha.sub.2
-globulin and beta-globulin. Goodger (1970, ibid) also reported that on
slab agarose electrophoresis, bovine hapteglobin migrated to the
beta-globulin region. Liberg (ibid) reported a significant increase in the
number of serum protein bands found in the region near alpha.sub.2
-globulin and beta-globulin in animals with signs of inflammation. This
information supports the conclusion that the single immunoprecipitation
band seen in this region of lanes 3 and 6 (TPR), but not in lane 4 (CPR),
when the gel is probed with Hap1 Mab is, in fact, haptoglobin.
EXAMPLE 3
Haptoglobin Assay
As noted in Example 1, the day 0 and day 4 post-treatment plasma (TP)
samples were assayed using the ELISA of Example 2 to determine bovine
haptoglobin levels.
FIG. 5 shows a typical standard curve obtained with the ELISA. The mg %
HbBC of haptoglobin in the gelatins was determined by the differential
peroxidase activity method and plotted against the ELISA O.D. reading
taken at 405 nm.
Shown in Table 1 are the haptoglobin levels (mg % HbBC) of plasma samples
from 4 cows taken at the two timepoints, one immediately prior to
treatment and the second at 4 days post-treatment. The haptoglobin levels
were determined both by ELISA and by the differential peroxidase method.
This data clearly shows an increase in haptoglobin levels in the test
animals at day 4 post-treatment while no corresponding increase is seen in
control animals. Additionally, the haptoglobin levels obtained by the
ELISA method are comparable to those obtained by the differential
peroxidase method.
It is understood that the foregoing detailed description is given merely by
way of illustration and that modifications and variations may be made
therein without departing from the spirit and scope of the invention.
TABLE 1
______________________________________
Haptoglobin Levels (mg % HbBC)
Day 0 Day 4
Animal # ELISA.sup.a
Peroxidase.sup.b
ELISA Peroxidase
______________________________________
Test-1.sup.c
0 0 40 32
Test 2 0 0 41 56
Control-1.sup.d
0 0 0 0
Control-2
0 0 7 0
______________________________________
.sup.a Haptoglobin levels measured by extrapolation to standard curve
shown in FIG. 5
.sup.b Haptoglobin levels measured by differential peroxidase method
.sup.c Test cows were injected with Turpentine as described in methods
.sup.d Control cows were injected with Normal Saline as described in
methods
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